PROJECT SUMMARY Cerebrospinal fluid (CSF) leak is a common complication of numerous procedures in otolaryngology. It has been estimated that up to 13.8% of endoscopic skull base surgeries result in CSF leaks. In the acute setting, imaging techniques such as magnetic resonance imaging (MRI) or computer tomography (CT) are used for assessment. Patients with high enough clinical suspicion may be taken directly to the operating room for management, which involves identifying the site of the leak and using either native tissue or biocompatible materials to patch the affected site. It is often difficult, however, to detect CSF leaks in clinical or postoperative hospital settings, as it is not unusual for postoperative patients to have secretions, and therefore distinguishing normal secretions from those containing CSF can be challenging. Failure to recognize and repair a leak can result in severe complications, such as meningitis, brainstem herniation, and death. Currently, there are no proven, available tests that allow a physician, who is concerned about a CSF leak, to cheaply and non- invasively rule out the presence of a leak. As a result, physicians must return to the same diagnostic modalities that are used in the acute setting. However, the costly and invasive nature of these diagnostic tests make them difficult to justify in a patient who seems otherwise well. Alternative methods have been developed for the detection of CSF leaks, such as beta-2 transferrin electrophoresis or enzyme-linked immunosorbent assay (ELISA); however, they are rarely used due to high cost and long time-to-result. More recently, researchers have looked into the detection of beta-trace protein (?TP) and found it to be comparable to beta-2 transferrin in sensitivity and specificity for CSF. Although used in a research setting, this protein has yet to be used for clinical detection. The main goal of this project is to develop a next generation, rapid, inexpensive and simple diagnostic device for detection of CSF leaks. The device will incorporate a sample pre-concentration step using the aqueous two- phase system (ATPS), colorimetric enzymes for signal amplification, and a rapid lateral-flow immunoassay (LFA) for detection. The traditional LFA is not sensitive enough for the detection of ?TP at the relevant concentrations. To overcome this barrier, the ATPS can be used to concentrate the target protein by several orders of magnitude prior to LFA detection. By using paper microfluidics, we have demonstrated that our device can simultaneously and seamlessly concentrate and detect target proteins. To improve the LFA detection limit even further, we have demonstrated the feasibility of using the ATPS as a novel method of sequentially delivering signal enhancement reagents across a detection zone. We will develop the prototypes of our device using two approaches: a ?low-hanging fruit? two-stage platform, and a ?high-risk, high-reward? one-stage platform. Once fully developed, the device will allow clinicians to more rapidly detect and treat CSF leaks, as well as be used to prevent patients who otherwise appear well from receiving expensive and invasive studies and procedures. Furthermore, such a device is not limited to the field of otolaryngology, but may also find use in identifying injuries to the spinal cord or globe, and may have a role in ruling out a CSF leak in postoperative neurosurgical patients with a low pretest probability of having a leak.